Akusu, O.M et al., IJFNR, 2019; 3:20

Research Article IJFNR (2019) 3:20

International Journal of Food and Nutrition Research (ISSN:2572-8784)

Changes in Microbiological Quality of Table Spreads Produced from African Pear () Pulp during Storage Akusu, Ohwesiri Monday*; Wordu, Gabriel Oji and Obiesie Chidiebere

Department of Food Science and Technology, Rivers State University, Nkpolu Oroworukwo, P. M. B., 5080, Port Harcourt, Nigeria

ABSTRACT African pear (Dacryodes edulis) pulp was extracted and *Correspondence to Author: pasteurized. The pasteurized pulp was homogenized with Akusu, Ohwesiri Monday different levels of food grade additives to form table spreads of Department of Food Science and samples A to H while sample I was left without preservative. Technology, Rivers State Universi- The spreads were packed in sealed glass containers and ty, Nkpolu Oroworukwo, P. M. B., stored at room temperature (28±20C) for 4 weeks to evaluate the changes in microbiological quality of table spread during 5080, Port Harcourt, Nigeria storage period. Samples were collected in a weekly interval to study the microbiological assay of the spread starting from week zero to the last week. High total bacteria count of 1.8x107CFU/ How to cite this article: ml was seen in sample I (spread without preservative) at week Akusu O. M, Wordu G. O and zero, this increased significantly to 8.1x108CFU/ml after 3 weeks Obiesie C. Changes in of storage and TNTC (too numerous to count) after 4 weeks of storage at 28±20C. The least growth were observed in samples Microbiological Quality of Table A and C with bacteria counts of 8.1x107CFU/ml and 3.5x107CFU/ Spreads Produced from Af-rican ml, respectively. The least fungi count of 2.0x106CFU/ml was Pear (Dacryodes edulis) Pulp noted in sample C after 4 weeks of storage while the highest during Storage. International Jour- fungi count of 4.5x107CFU/ml was seen in sample I after 4 nal of Food and Nutrition Research, weeks of storage at room temperature (28±20C). The suspected 2019; 3:20. microorganisms based on their morphology were; E. coli, Staphylococcus aureus, Salmonella specie while fungi were Penicillium specie and Aspergillus specie. Deterioration sets in significantly after two weeks storage as total bacteria and fungi counts rose above 1.0x107 and 1.5x106, respectively. The microbiological quality of the samples was stable up to the eSciPub LLC, Houston, TX USA. second week of storage except sample I (without preservative). Website: http://escipub.com/

Keywords: Microbiological Quality, Vegetable Spreads, African Pear Pulp, Storage Temperature, Nutritional Quality, Preservatives

IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 1 Akusu, O.M et al., IJFNR, 2019; 3:20 1. Introduction pulp and its oil [14] and the oil extraction processes[15]. These studies revealed Food preservation is the science of extending excellent nutritional qualities of fruit pulp and the shelf life of food, maintaining its nutritional interesting food processing properties of the quality and avoiding the growth of unwanted oils extracted from the pulp and kernel safou microorganisms [1]. It is the process of treating [16]. These have also revealed the importance and handling food to stop or slow spoilage of this fruit nutritionally, therapeutically and in which results to loss of quality, edibility or cosmetics. The pulp, the only edible part of the nutritional values and thus allow for longer fruit is particularly rich in lipids, hence, it could storage. Preservation involves preventing the be an important source of oil [17]. Besides growth of bacteria, yeast, fungi and other lipids, D. edulis pulp contains substantial microorganisms [2]. Vegetable spreads are amount of many other nutrients including spreadable product having at least 90% proteins, carbohydrates, minerals, vitamins and ingredients from nuts and pulps used in various fibres[16]). forms, such as paste and slurry [3,4]. Vegetable spreads are used like the commercially High content of protein and other essential available butter, they can be produced from nutrients in African pear spread makes it almond, cashew, hazelnut, macadamia nut, susceptible to microbiological contamination peanut, pecan, pistachio and walnut [5]. and growth. Thus, the objective of this work Vegetable spread is popular and widely was to produce table spreads from African pear accepted by consumers due to its flavour, good pulp treated with different levels of nutritional values and suitability for preservatives and to evaluate the effect of room 0 consumption either alone or in combination with temperature (28±2 C) storage on the a variety of other foods. African pear pulp has microbiological quality of the product. great potential for vegetable spread production. 2. Materials and Method The African pear tree (Dacryodes edulis) is a 2.1. Materials tropical oleiferous fruit tree that possesses Mature and good quality fruits of the African enormous potential in Africa [6]. Various parts pear (Dacryodes edulis) were purchased from of the tree are used in traditional medicine[7,8]. the fruit market in Port Harcourt and transferred The serves for firewood and carpentry [9]. to the Microbiological Laboratory in the The entire tree is used in Agro-forestry systems Department of Food Science and Technology, for soil conservation [10]. Dacryodes edulis fruit Rivers State University, Port Harcourt, Nigeria. is popular in the diets of many Africans. It can All apparatus and chemicals used for the study be eaten raw, roasted or boiled in hot water and were obtained from the same Laboratory and is eaten alone or used in garnishing cooked or were of analytical grade. roasted maize. It could also be used as butter 2.2. Extraction of African Pear Pulp and the to eat bread [8]. It has been found in Cameroon Formulation of the Spread recipe that when the pulp of African pear is cooked and seasoned, it serves as spread The pulp was extracted using modified [11]). According to Ayuku et al.[12], D. edulis traditional method of pear roasting. African pear has a potential to improve nutrition and food fruits were sorted and washed with tap water security. During the last three decades, more and sodium chloride solution, furthermore 0 and more studies have been conducted on D. rinsed thoroughly. They were roasted at 60 C edulis, essentially the tree and its fruit. The for 4 min in a hot air oven (model QUB scientific researches on D. edulis focused on 305010G, Gallenkamp, UK). The roasted fruits the characterisation of propagation techniques were allowed to cool for 10 min, the thin bluish- of D. edulis tree [13], the nutritive value of its black epicarp were gently removed and

IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 2 Akusu, O.M et al., IJFNR, 2019; 3:20 discarded while the soft pulps were scraped off The mixture was homogenized properly using a and recovered in sterile stainless steel plates. laboratory stirrer (model JKL 2145, REMI The extracted pulp was pasteurized by heating Motors, India) to produce the African pear at 1000C for 5 min in a stainless pot. The spread (APS) as presented in Figure 1 and pasteurized pear pulp was treated with different Table 1. levels of recommended food preservatives [18].

African Pear (Dacryodes edulis) Fruit

Sorting/Cleaning

Oven roasting (600C for 4 min)

Cooling (10 min)

Peeling (Recovery of pulp from the hard central core)

Pasteurizing (1000C for 5 min)

Homogenizing (with addictives/preservatives)

African Pear Spread

Figure1. Flow Chart for the Production of African Pear Spread

Table 1. African Pear Spread (APS) Formulations

SAMPLES PEAR PULP H2O PS (E200) BHT (E321) CA (E330) (g) (g) (g) (g) (g) A 100 15 0.045 B 100 15 0.035 C 100 15 0.01 D 100 15 0.005 E 100 15 0.008 F 100 15 0.005 G 100 15 0.025 0.005 H 100 15 0.005 0.008 I 100 15 Key: PS = Potassium sorbate, BHT = Butylated hydroxytoluen, CA = Citric acid.

2.3. Storage of Samples from week one, two, three and four using week The produced spreads were stored in sealed zero as the control sample. glass containers at room temperature (28±20C). 2.4. Microbiological Analysis Samples were collected at one-week interval Storage stability of the spreads were conducted for a period of four weeks for storage studies to study the changes in microbiological quality which included; total bacterial count, total fungi

IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 3 Akusu, O.M et al., IJFNR, 2019; 3:20 count and an assay of the microorganisms using the spread plate (surface plating) method. associated with the spoilage of African pear The five tubes (10-5) were used for inoculation. spreads dusring storage. From each of these tubes, 0.1ml was pipette 2.4.1. Total Bacteria and Fungi Count onto the surface of an agar plate and spread evenly using a sterile L-shaped glass spreader. Microbial assay was carried out on each of the The plates were incubated at 350C for 24 - 48 stored spread samples to test the presence of hr for NA and 300C for 96 hr for PDA. The bacteria and fungi using nutrient agar (NA) and colonies were counted using a colony counter potato dextrose agar (PDA) for total bacteria and result recorded as colony forming unit per and fungi counts, respectively. The procedure ml (CFU/ml). described by the International Commission on Microbiological Specification for Food [19] was 2.4.5. Isolation and Identification of adopted in this study. Microorganisms Associated with the Spoilage 2.4.2. Sterilization of Materials This was done through sub-culture to obtain All glassware used were sterilized by pure culture of isolates. Single colony of autoclaving at 121oC for 15 min and allowed to bacteria was randomly isolated from the mother cool before use. culture onto a fresh prepared nutrient agar 2.4.3. Culture Media Preparation plate. It was inoculated using streak method The powder media were weighed and dissolved and incubated at 370C for 24 hr for proper in distilled water in accordance to each dilution identification. factor as follows: NA: 28g powder in 1000ml 2.4.6. Gram Staining distilled water to obtain 2.8% (w/v) solution. A loop full of fresh culture was isolated and PDA: 39g powder in 1000ml distilled water to emulsified in a drop of distilled water and obtain 3.9% (w/v) solution. The mixtures were allowed to air-dry. The film was heat-fixed by stirred separately, properly and sterilized at passing it over a flame for 3 sec before 1210C for 15 min in an autoclave. They were covering with crystal violet for 60 sec. It was allowed to cool to 450C and then dispensed into rinsed with running tap water, drained and dishes using the spread-plate technique. covered with iodine for 60 sec, rinsed again For diluents' preparation, one tablet of with water and decolorized with ethanol for 30 phosphate buffer was dissolved in 100ml of sec, counterstained with safrain for 60 sec after distilled water. Using a 10ml syringe, 9ml each rinsing off the ethanol. The film was rinsed of the buffer solution were injected into five (5) finally and air-dried before viewing under the separate test tubes. Each of the tubes were microscope. covered with cotton wool and aluminium foil 2.4.7. Identification of Isolates and then, sterilized at 1210C for 15 min in the Autoclave. The following characteristics were used for identification of the isolates: size of colony, 2.4.4. Serial Dilution and Inoculation shape, edge, elevation, pigmentation, texture, The spread sample was weighed (1g) and opacity, consistency and odour of colony. suspended in the first test tube (containing 9ml 2.4.8. Biochemical Test diluents), making the solution 10ml. Serial dilution was done; by pipetting 1ml from the The following biochemical test were done for primary suspension (10-1) into the next tube identification and confirmation of the isolates: (10-2), 1ml from that 2nd tube into the next, the indole test, motility, oxidase, catalase, methyl process continued to the last (fifth) tube. One red and voges proskeur test. ml from the last tube was removed and 3. Results and Discussion discarded. Prepared plates were inoculated 3.1. Total Bacteria and Fungi Counts IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 4 Akusu, O.M et al., IJFNR, 2019; 3:20 As shown in Table 2, samples A, B, C and F did also showed no growth at week 1 as presented not show any bacteria growth at week zero. in Table 3. The least fungi count of High total bacteria count of 1.8x107CFU/ml was 2.0x106CFU/ml was noted in sample C while seen in sample I (spread without preservative) the highest fungi count of 4.5x107CFU/ml was at week zero, this increased significantly to observed in sample I after 4 weeks of storage 8.1x108CFU/ml after 3 weeks of storage and period at room temperature (28±20C). This TNTC (too numerous to count) after 4 weeks of implies that BHT preservative was effective in storage at 28±20C. The least growth at week 4 subduing the bacterial and fungal growth in the were recorded in samples A and C with produced pear spread. The total plate count of bacteria counts of 8.1x107CFU/ml and all the spread samples were beyond the 3.5x107CFU/ml, respectively. marginal acceptable count of 5.0 x 106CFU/ml, Fungal growth was not observed in all the for table spreads and margarine [19]. samples at week zero. Also Samples C and F

Table 2. Total Bacteria Counts (CFU/ml) on APS Treated with Different Levels of Preservatives during Storage Samples Storage Time (Weeks) 0 1 2 3 4 A NG 1.6 x 106 1.9 x 107 3.5 x 107 8.1 x 107 B NG 1.0 x 106 1.6 x 107 3.3 x 107 1.0 x 108 C NG 1.0 x 106 3.0 x 106 1.0 x 107 3.5 x 107 D 6.0 x 106 2.8 x 107 4.6 x 107 2.0 x 108 6.1 x 108 E 1.2 x 106 1.0 x 107 4.9 x 107 8.0 x107 1.7 x 108 F NG 2.0 x 106 5.6 x 106 1.2 x 107 1.0 x 108 G 1.0 x106 6.0 x 10b 2.9 x 107 1.1 x 108 2.0 x 108 H 3.0 x 106 1.9 x 107 2.9 x 107 8.1 x 107 1.5 x 108 I 1.8 x 107 4.7 x 107 8.9 x 107 8.1 x 108 TNTC

Table 3. Total Fungi Counts (CFU/ml) on APS Treated with Different Levels of Preservatives during Storage Samples Storage Time (Weeks) 0 1 2 3 4 A NG 2.0 x 106 2.0 x 106 3.0 x 106 3.5 x 106 B NG 1.1 X 106 2.0 x 106 2.8 x 106 3.0 x 106 C NG NG 1.0 x 106 1.5 x106 2.0 x 106 D NG 1.0 x106 1.0 x 106 2.0 x 106 1.1 x 107 E NG 2.0 x 106 2.0 x 106 8.0 x 106 1.7 x 107 F NG NG 1.0 x 106 2.0 x 106 3.0 x 106 G NG 2.0 x 106 3.0 x 106 8.0 x 106 1.4 x 107 H NG 1.0 x 106 1.1 x 106 2.0 x 106 1.0 x 107 I NG 2.0 x 106 2.0 x 106 1.7 x 107 4.5 x 107

Key: APS = African pear spread; A = APS treated with potassium sorbate (0.045%);B = APS treated with potassium sorbate (0.035%); C = APS treated with BHT (0.01%);D = APS treated with BHT (0.005%);E = APS treated with citric acid (0.008%);F = APS treated with citric acid (0.005%); G = APS treated with BHT (0.005%) and potassium sorbate (0.025%); H = APS treated with citric acid (0.008%) and BHT (0.005%); I = APS without preservative; NG = no growth; TNTC = too numerous to count

IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 5 Akusu, O.M et al., IJFNR, 2019; 3:20 3.2. Microorganisms Associated with OX positive (+) and catalase positive (+). Spoilage of APS and their Biochemical Salmonella specie which was Gram negative (- Reactions ), VP negative (-), Ind negative (-), OX negative The suspected microorganisms based on their (-) with positive (+) motility. Penicillium specie morphology were; E. coli which was Gram which its colonies grew in shades of green with negative (-), VP negative (-), Ind negative (-), broom-like head. Aspergillus specie, its MR negative (-), OX negative (-), with positive colonies also grew in shades of yellowish-green (+) motility as shown in Table 4. with swollen vesicle and flasked-shaped Staphylococcus aureus which was Gram phialides. positive (+), VP negative (-), Ind negative (-),

Table 4. Microorganisms Associated with Spoilage of APS and their Biochemical Reactions

Suspected Organism Gram VP Ind. MR Motility OX Catalase RXN

E. Coli - - - + - .

Staph. Aureus + - - + +

Salinonella sp - - - + -

Key:VP = voges proskeur, Ind = Indole, MR = methyl red, OX = oxidase,

4. Conclusion Peanut Butter; United State Department of Agriculture, Washington, DC, USA. The findings from this work showed that table 4. Nielsen SS (2010). Food Analysis; Springer: New spreads produced from African pear pulp and York, NY, USA. treated with 0.010% BHT preserved better than 5. Mangels R (2001). Guide to nuts and nut butters. citric acid and potassium sorbate. The least Vegetarian Journal, 21, 20-23. bacteria counts were observed in samples A 6. Kengué J (1990). Le Safoutier (Dacryodes edulis (G.Don) H.J.Lam), premières données sur la and C. In terms of fungi, least and highest morphologie et la biologie. Thèse de doctorat, counts were recorded in samples C and I, Université de Yaoundé, Cameroun. respectively after 4 weeks of storage period at 7. Okafor JC (1983). Varietal delimitation in room temperature (28±20C). The Dacryodes edulis (G.Don) H.J.lam microbiological quality of the table spreads (). International Tree Crops Journal, 2, 255-265. treated with food grade preservatives was 8. Duru M, Amadi C, Ugbogu A, Eze A and Amadi stable up to the second week of storage. The B (2012). Phytochemical, vitamin and proximate need to review the treatment content with composition of Dacryodes edulis fruit at different cognisance to good manufacturing practice stages of maturation. Asian Journal of (GMP) is highly recommended. Science and Research, 2(4), 437-441. 9. Ndoye O, Ruiz-Pérez M and Eyebe A (1997). References The Markets of Non-Timber Forest Products in 1. Rahman SM (2007). Hand Book of Food the Humid Forest Zone of Cameroon. ODI Rural Preservation. 2nd Edn. London. Taylor and Development Forestry Network Paper, Francis Group. Yaounde, Cameroon. 2. FAO (2005). Food and Agriculture Organization 10. Ndangang V (1989). A Survey of Traditional of the United Nations. Principles and practices of Agro-Forestry Woody of the North-West small- and medium-scale fruit juice processing. Province of Cameroon. Pre-Optional Available:http://www.fao.org/docrep/005/y2 Study Report, National Advance School, 515e/y2515e09.htm Agriculture, Yaounde, Cameroon, p. 72. 3. USDA (2006). United States Department of 11. Kadji BRL, Kone FMT, Sika AE and Dabonne SI Agriculture. Commercial Item Description. (2016). Physico-chemical properties of Safou IJFNR: http://escipub.com/international-journal-of-food-nutrition-research/ 6 Akusu, O.M et al., IJFNR, 2019; 3:20 (Dacryodes edulis) fruits grown in Côte d’Ivoire. Journal of Applied Biosciences, 105, 10103- 10110. 12. Ayuku ET, Duguma B, Kengue J, Tiki–managa T and Zekkeng P (2000). Uses, management and economic potential of Dacryodes edulis in human low-land Cameroon. Economic Botany, 53(3), 292-300. 13. Kengué J (2002). Fruits for the Future 3. Safou: Dacryodes edulis. Research for Development in Department for International Development. p. 147. 14. Ajayi IA, Oderinde RA, Kajogbola DO and Uponi JI (2006). Oil content and fatty acid composition of some underutilized legumes from Nigeria. Food Chemistry, 99(1), 115-120. 15. Kapseu C (2009). Production, analyse et applications des huiles végétales en Afrique. Oleagineux Corps Gras, Lipides, 16(4), 215-229. 16. Poligui RN, Mouaragadja I, Haubruge E and Francis F. (2013). La culture du safoutier (Dacryodes edulis (G. Don) H.J.Lam (Burseraceae)): enjeux et perspectives de valorisation au Gabon (synthèse bibliographique). Biotechnologie Agronomie société et Environnement, 17(1), 131-147. 17. Ondo-Azi A, Ella Missang M, Silou T and Chalchat J (2013). Variation in Physicochemical Characteristics of Safou (Dacryodes edulis (G. Don) H.J. Lam) fruits. 18. CODEX-STAN (1999). CODEX Alimentairus Commission Standard for Named Vegetable Oils, FAO Corporate Document, CODEX STAN 210. Retrieved from https://www.fao.org/docrep/004/y2774e05. 5th July, 2016. 19. ICMSF (1995). International Commission On Microbiological Specification for Foods. Food Administration Manual. Version 2.0. Retrieved from http://www.icmsf.org. 3rd July, 2017).

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